We continue to investigate the role of membrane CPE and secretogranin III as sorting receptors for targeting POMC to the regulated secretory pathway (RSP). CPE knockout (KO) mice show defective trafficking of POMC in primary cultures of anterior pituitary cells since 50% of the newly synthesized POMC was targeted for degradation in the absence of CPE. However, some of the remaining POMC was sorted into the RSP, presumably by Secretogranin III (SgIII). SgIII is found in neuroendocrine cells and is involved in trafficking of chromogranin A (CgA) to the RSP. Our studies using RNA interference to knock down SgIII and CPE showed that both proteins affect the normal secretion behavior of POMC in AtT20 cells, i.e. POMC was secreted at an elevated rate through the constitutive secretory pathway when either CPE or SgIII are reduced. When both are knocked-down, the affect is augmented, suggesting that POMC trafficking is dependent on both proteins for efficient trafficking to the RSP. In other studies of cellular secretion pathways, and in collaboration with Dr. Bruce Baum (NIDCR), we generated a proinsulin mutant (B10-proinsulin) and mutants of human growth hormone (hGH) and expressed them in mouse submandibular glands (SG). These glands provide a delivery route to either the saliva or the circulation. In the case of B10-proinsulin, significant amounts were secreted into the circulation where it functioned to alleviate hyperglycemia in chemically induced diabetic mice. Additionally, we identified a hGH mutant that was secreted into the circulation, in contrast to the WT hGH control, that was secreted almost exclusively into the saliva. This suggests that the SG have the potential to be a target tissue for gene therapy and that the knowledge of sorting determinants of peptide hormones will play a crucial part in this process. In collaboration with Dr. Bruno Tota (University of Calabria), pGlu-serpinin was found to have positive inotropic activity in cardiac function, with no change in blood pressure and heart rate. pGlu-serpinin acts through a beta1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway and emerges as a novel-adrenergic inotropic and lusitropic modulator of the myocardium in response to sympathochromaffin stimulation. We have also investigated the effect of pGlu-serpinin on cardioprotection. Using normotensive (WKY) and hypertensive (SHR) rats as models; we showed that pGlu-serpinin mimicked pre-conditioning and post-conditioning-induced cardioprotection. In both WKY and SHR rats, pGlu-serpinin improved left ventricle function recovery after ischemia. Moreover, it reduced ischemicinduced contracture state and decreased infarct size. In pGlu-serpinin mediated post-conditioning pharmacological cardiac protection, the mechanism involved the activation of the reperfusion injury salvage kinase (RISK) pathway. CPE plays a significant role in obesity and obese CPE-KO mice have low bone mineral density. We showed that the lack of processing of pro-CART to mature CART, a peptide that promotes bone formation, contributes significantly to the poor bone density in these mice. Additionally in collaboration with Dr. Lecka-Czernik (Univ. of Toledo), we found that CPE is enriched in a rat messenchymal stem cell line from bone marrow and thus CPE may be involved in regulating bone formation at another level. We found that primary cultures of mouse mesenchymal stem cells that normally differentiate into osteoblasts could be partially switched to a cell type that accumulated lipid when incubated with purified CPE protein. This represents a novel discovery that CPE may contribute to adipogenesis in this pathway. CPE-KO mice have deficiencies in their nervous system function, including learning and memory. This defect is attributed to the loss of neurons in the CA3 region of the hippocampus due to the physical stress of ear tagging and tail clipping for genotyping and the emotional stress of maternal separation at the time of weaning (3 weeks). The neurodegeneration was prevented by the drug carbamazepine, and anti-epileptic drug, demonstrating that the stress associated with weaning induced epileptic-like seizures resulting in neuronal cell death in the absence of CPE. We also examined the effect of restraint stress on CPE expression in hippocampal neurons. When mice were subjected to mild chronic stress (1h/day for 7days), which increases glucocorticoid secretion, the mice showed an increase in CPE mRNA and protein in the hippocampus, and no neuronal degeneration was evident. Furthermore, when hippocampal neurons were treated with synthetic glucocorticoid, dexamethasone, there was a significant increase in CPE mRNA and protein in the cells. These observations suggest that the increase in CPE may mediate neuronal survival during stress and lack of CPE results in neuronal degeneration. To this end, we have applied recombinant CPE to rat hippocampal neurons in culture and shown increased survival and neuroprotective effect of CPE on these neurons when subjected to oxidative stress with hydrogen peroxide treatment or glutamate cytotoxicity. Since CPE can act as a neuroprotective molecule with trophic properties, we have recently named it neurotrophic factor-alpha1 (NF-alpha1) to indicate this when CPE functions as a trophic factor. Recently we have investigated the role of CPE in restraint stress-induced depression. Prolonged, but not short-term, stress reduces fibroblast growth factor 2 (FGF2) in the hippocampus, leading to depressive-like behavior in mice. We show in cultured hippocampal neurons that exogenous carboxypeptidase E (CPE/NF-alpha1) up-regulated FGF2 expression independent of its enzymatic activity. In vivo, we found that mice after short-term restraint stress increased hippocampal CPE, FGF2 and doublecortin prior to depressive-like behavior onset. CPE-KO mice exhibited severely reduced hippocampal FGF2 levels and immature neuron numbers in the subgranular zone. These mice displayed depressive-like behavior that was rescued by FGF2 administration. Thus, CPE appears to up-regulate hippocampal FGF2 expression during stress, which leads anti-depressant effects. The expression of CPE was examined in mouse embryos to determine if it could play a role in early embryonic development. We found that WT CPE and CPE-deltaN mRNA was expressed as early as day E5.5 and increased each day, peaking at E8.5, falling slightly at E9.5 prior to expression of the endocrine system. CPE mRNA expression decreased sharply at E 10.5-11.5 to below E5.5 levels and then increased sharply at E12.5 in parallel with the development of the endocrine system and continued to increase to adulthood. However, CPE-deltaN mRNA increased maximally at E10.5 followed by a precipitous decrease at E11.5-12.5, and then a small increase till PN1. In contrast to CPE, CPE-deltaN is absent in the adult hippocampus. In situ hybridization studies indicate that WT CPE and CPEdeltaN mRNA are expressed primarily in the fore brain and somites in mouse embryos. We have begun to study the role of CPE during embryonic development of the nervous system using neurospheres to study proliferation and differentiation. Exogenous addition of recombinant CPE to E13.5 neocortex-derived neurospheres, reduced proliferation of the neurospheres without causing cell death. Furthermore, neurospheres from 7d cultures that were dissociated into single cells and cultured for an additional 5d showed an increase in astrocytes in the presence of CPE, without altering the percentage of neuronal and oligodendrocyte populations. An enzymatically inactive mutant form of CPE was also able to drive differentiation to astrocytes indicating that the enzymatic function of CPE is not essential. Our results suggest a novel role of CPE as an extracellular signal to differentiate neural stem cells into astrocytes.